The vitamin D endocrine system is fundamental to health maintenance and disease prevention. Bone homeostasis and repair is regulated by a network of vitamin D metabolites, of which 1,25-dihydroxyvitamin D (1,25OHD) is considered the most biologically active and relevant in bone health. Vitamin D is also metabolized into other forms, including 24,25-dihydroxyvitamin D (24,25OHD), which we believe is fundamental for human mesenchymal stem cell (hMSC) osteogenic maturation and bone formation. Our data establishes that 24,25OHD inhibits proliferation, increases alkaline phosphatase activity and mineralization, and decreases 1-hydroxylase expression, potentially decreasing the ability of hMSC to convert 25OHD to 1,25OHD, while promoting osteogenic maturation. hMSC also express a 24,25OHD receptor (24,25OHDR), suggesting direct actions of 24,25OHD, and further implicating 24,25OHD in osteogenic maturation. Currently, we do not understand how 24,25OHD and 1,25OHD differentially mediate this process. During murine development, the transcription factor p63 is required for skeletal formation, and our recent work demonstrated that p63 is integral for hepatocyte growth factor (HGF) and 1,25OHD induced VDR expression and osteogenic maturation. This demonstrates that p63 mediates 1,25OHD/VDR, and poses the question as to its regulation of 24,25OHD actions. p63 gene expression is complex, leading to the formation of TA- and Np63 isoforms, and three splice variants (,,?). The TA/Np63 isoforms act in opposition to activate or repress growth and differentiation, however the biological significance of p63 splice variants (,,?) in osteogenic maturation and bone repair are not established. Our studies demonstrate up-regulation of Np63 isoforms by 1,25OHD and alteration of p63? by 24,25OHD, linking 1,25OHD up-regulation of Np63, required for terminal differentiation, and 24,25OHD up-regulation of p63, known to regulate VDR expression. Thus, p63 appears to be central in the modulation of bone homeostasis by 1,25OHD and 24,25OHD. We hypothesize that the actions of vitamin D on osteogenic maturation and bone repair are due to a regulatory relationship between p63 gene products and unique 24,25OHD and 1,25OHD effects. We will test this hypothesis by assessing the regulation of p63 gene products by 24,25OHD and 1,25OHD. We expect Np63 will increase with 1,25OHD, and 24,25OHD will increase the p63? variants. To confirm the requirement of p63 during this process, we will use stable shRNA lentiviral vectors to knock-down first the TA- and Np63 forms, followed by the splice variants (,,?) . We predict that ablation of Np63? will block vitamin D induced effects. To test this hypothesis in vivo, we will use a rat xenograft model of bone repair. We predict that over-expression of Np63? will increase the osteogenic maturation of hMSC, promoting bone repair. We expect to identify a variant of p63 as a major regulatory component of osteogenic maturation by 1,25OHD and 24,25OHD. Those results would support a generalized paradigm implicating p63 as a key player during hMSC differentiation and bone repair.